Soluble and immunoreactive zika virus ns1 polypeptides

ABSTRACT

The disclosure concerns a polypeptide suitable for detecting antibodies against Zika virus in an isolated biological sample having a Zika virus NS1 wing domain specific amino acid sequence and variants thereof, wherein no further Zika virus specific amino acid sequences are present in the polypeptide. This polypeptide does not immunologically cross-react with antibodies raised against structurally related antigens from tick-borne encephalitis virus, Dengue virus 1-4, West Nile virus, yellow fever virus or Japanese encephalitis virus, but immunologically reacts with antibodies raised against full length Zika virus NS1 antigen. Also disclosed is a method of producing a soluble and immunoreactive Zika virus NS1 polypeptide as well as methods and kits for detecting antibodies specific for Zika virus in an isolated sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/EP2018/060330 filed Apr. 23, 2018, which claims priority to EuropeanApplication No. 17168197.6 filed Apr. 26, 2017, the disclosures of whichare hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Zika virus is an arbovirus (arthropod-borne virus, i.e. virustransmitted by arthropod vectors such as insects) that belongs to theFlaviviridae family of viruses and is spread by the Egyptian and theAsian mosquito strains Aedes aegypti and Aedes albopictus. In the years2013 and 2014, the incidence of Zika infections in French Polynesiaescalated, followed by large outbreaks in Latin America since 2015,peaking epidemic-like at the time around the Olympic Games in Brazil in2016. So far, efficacious medical treatment or vaccination is lacking.In healthy persons an infection with Zika virus usually leads to no oronly mild symptoms. However, for pregnant women the situation isdifferent. The Zika virus can be transmitted from a pregnant woman toher unborn child and is suspected to cause severe brain malformationsand defects such as microcephaly. Microcephaly (derived from Greek for“small head”) is a condition in which a baby's brain does not developproperly, and thus its head has a smaller size than normal. Infection inadults may lead to the so-called Guillan-Barré syndrome (muscle weaknesscaused by the immune system which attacks the peripheral nervoussystem). Efficacious medical treatment after infection is lacking. Inorder to receive timely supportive treatment it is crucial to know if apatient has been infected with Zika virus. In particular, highlyspecific immunoassays are utterly needed in regions with high prevalenceof multiple flaviviral infections. As a matter of course, it is adaunting task to reliably diagnose, e.g., a recent Zika infection in anindividual that has undergone other flaviviral infections such as Dengueor yellow fever in the past and whose serum is characterized bypolyclonal antibodies against the main immunogens of Dengue and yellowfever virus.

Therefore, apart from a vaccination and medication development strategy,also highly sensitive and specific serological diagnostics need to bedeveloped in order to reliably confirm or rule out an infection of apatient with Zika virus. Since 2016 a couple of ELISA-based immunoassaysdetecting antibodies against the Zika virus NS1 antigen are commerciallyavailable (e.g. Huzly et al., Euro Surveill. 2016; 21 (16) pii=30203,1-4). However, these assays are suspected to cross-react with antibodiesthat have been raised against related viruses such as Dengue virus andother arboviruses that belong to the family of flaviviruses like WestNile virus, yellow fever virus, tick-borne encephalitis virus (FSME) orJapanese encephalitis virus. This cross-reactivity would lead to falsepositive results and erroneous interpretation of a patient's immunestatus and seems to be due to the structural and sequence homologies ofZika NS1 (non-structural antigen) and its counterparts in otherflaviviruses such as West Nile and Dengue viruses (Hilgenfeld 27 Oct.2016, Embo J. 1-3).

It is conceivable that the incidence and prevalence data that have beenreported for the Zika epidemic in Brazil in 2015/2016, have suffered theflaw of an exaggeration bias due to the limited specificity of theimmunoassays that have been used at the time. In order to evaluateprevalence and incidence data and to assess the objective risk of anemerging epidemic, clear-cut diagnostics are an indispensableprerequisite. Serologic assays with poor specificity would overemphasizethe extent of an epidemic and therefore lead to panic-driven decisionsnot only by the authorities, but also by unsettled and frightenedindividuals. For instance, it has been reported that the number ofabortions in Brazil has significantly increased in the wake of themedial reporting on the true or ostensible Zika epidemic.

The crystal structure of full-length, glycosylated NS1 from West Nileand Dengue virus has been solved at high resolution and reveals distinctdomains and a rather complex protein topology (Akey et al., Science(2014; 343 (6173): 881-885). Recently, the crystal structure of aC-terminal fragment (amino acid residues 172-352) of the Zika virusnonstructural protein 1 (NS1) has been published, revealing ahead-to-head dimer and confirming the oligomeric character of NS1 (Songet al. Nature Struct. Mol. Biol. 2016 (23) 5, 456-459). In addition, thecomplete three-dimensional structure of full-length Zika virus NS1 hasbeen published by Brown et al. Nature Struct. Mol. Biol. 2016 (23) 9,865-868, describing the NS1 structure in further detail. Thus, NS1 turnsout to be a very intricate and complex protein due to its oligomericstate, its glycosylation pattern and its abundance in cysteine residues.Publications on dedicated Zika antigens for diagnostic purposes aretherefore scarce, and reliable information on linear or conformationalB-cell epitopes that are unique in Zika antigens and would allow for aclear-cut discrimination between Zika virus and related flaviviruseshave not been available so far.

International patent applications WO2017/144173 and WO2017/144173describe variants of the full length Zika NS1 antigen of 352 aminoacids, listing several essential epitopes, wherein a ß-ladder domainepitope (positions 322 to 326) in the C-terminal part of the NS1 antigenis regarded as essential for reactivity.

The problem underlying the invention therefore is the limitedspecificity of the hitherto available immunoassays detecting antibodiesagainst Zika virus. The problem is solved by the current invention asspecified in the claims.

SUMMARY OF THE INVENTION

The invention relates to a polypeptide, and its use in immunoassays,that is suitable for detecting antibodies against Zika virus in anisolated biological sample comprising a Zika virus NS1 domain specificamino acid sequence and variants thereof, wherein no amino acidsequences from the Zika virus NS1 ß-ladder domain are present in saidpolypeptide. In an embodiment said Zika virus NS1 domain specific aminoacid sequence consists essentially of SEQ ID NO. 1 or 2. In anotherembodiment, no further Zika virus specific amino acid sequences arepresent in said polypeptide. This polypeptide does not immunologicallycross-react with antibodies raised against structurally related antigensfrom tick-borne encephalitis virus comprising NS1 polypeptides of Denguevirus 1-4, West Nile virus, yellow fever virus or Japanese encephalitisvirus, but immunologically reacts with antibodies raised against fulllength Zika virus NS1 antigen according to SEQ ID NO:3.

In an embodiment, this polypeptide does not immunologically cross-reactwith antibodies raised against structurally related antigens fromtick-borne encephalitis virus comprising any of SEQ ID NOs:5 or 6,and/or from Dengue virus 1-4 comprising any of SEQ ID NOs:7 to 14,and/or from West Nile virus comprising any of SEQ ID NOs:15 or 16,and/or from yellow fever virus comprising any of SEQ ID NOs:17 or 18,and/or from Japanese encephalitis virus comprising any of SEQ ID NOs:19to 20, but immunologically reacts with antibodies raised against fulllength Zika virus NS1 antigen according to SEQ ID NO:3.

Also disclosed is a method of producing a soluble and immunoreactiveZika virus NS1 polypeptide as well as methods for detecting antibodiesspecific for Zika virus in an isolated sample using a Zika virus NS1polypeptide as described above as binding partner for anti-Zika virusantibodies. These methods detect either IgG or IgM class antibodies orboth. Further disclosed is the use of said NS1 Zika virus polypeptide inan in vitro diagnostic test for detecting anti-Zika virus antibodies.

Further aspects are reagent kits for the detection of anti-Zika virusantibodies, comprising a Zika virus polypeptide as specified before.

Another aspect is a method for detecting antibodies against a Zika virusin an isolated biological sample presumed to contain antibodies againstat least one other non-Zika flavivirus, by using a Zika virus NS1polypeptide comprising a complete or partial sequence of the ß-ladderdomain as specific binding partner, wherein the cross-reactivity againstsaid non-Zika flavivirus is eliminated by adding a polypeptidecomprising the NS1 ß-ladder domain of said Zika virus in an unlabeledform, in an embodiment adding said polypeptide comprising the ß-ladderdomain as a quencher.

Legend to the Disclosed Amino Acid Sequences

The mature NS1 protein comprises 352 amino acid residues (NS1, 1-352).Within the NS1 protein, the wing domain comprises 151 amino acidresidues and spans the NS1 amino acid region 30-180. Thus, the wingdomain positions (1-151) easily translate into the NS1 numbering byadding 29 amino acid positions. Vice versa, the NS1 amino acid positionseasily translate into wing domain numbering by subtracting as many as 29amino acid positions; aa 1 (wing)=aa 30 (NS1), aa 2 (wing)=aa 31 (NS1),aa 3 (wing)=aa 32 (NS1) and so forth. In an analogous way the ß-ladderdomain positions (1-162) easily translate into the NS1 numbering byadding 190 amino acid positions as the NS1 positions 191-352 correspondto the ß-ladder domain.

SEQ ID NO: 1 Zika virus NS1 wing domain aa 30-180 with position 179 X =A or S or CDRYKYHPDSP RRLAAAVKQA WEEGICGISS VSRMENIMWK SVEGELNAIL EENGVQLTVV VGSVKNPMWRGPQRLPVPVN ELPHGWKAWG KSYFVRAAKT NNSFVVDGDT LKECPLKHRA WNSFLVEDHG FGVFHTSVWLKVREDYSLE X  D,SEQ ID NO: 2 Zika virus NS1 wing domain aa30-180 with C55, C143, C179ADRYKYHPDSP RRLAAAVKQA WEEGICGISS VSRMENIMWK SVEGELNAIL EENGVQLTVV VGSVKNPMWRGPQRLPVPVN ELPHGWKAWG KSYFVRAAKT NNSFVVDGDT LKECPLKHRA WNSFLVEDHG FGVFHTSVWLKVREDYSLEA D,SEQ ID NO: 3 Zika virus NS1 full length aa 1-352; this sequence isalso published as strain MR 766, UniProt ID W8Q7Q3; the correspondingnumbering within the full length Zika precursor polyprotein is aa795-1146DVGCSVDFSK RETRCGTGVF IYNDVEAWRD RYKYHPDSPR RLAAAVKQAW EEGICGISSVSRMENIMWKS VEGELNAILE ENGVQLTVVV GSVKNPMWRG PQRLPVPVNG LPHGWKAWGKSYFVRAAKTN NSFVVDGDTL KECPLKHRAW NSFLVEDHGF GVFHTSVWLK VREDYSLECDPAVIGTAVKG REAAHSDLGY WIESEKNDTW RLKRAHLIEM KTCEWPKSHT LWTDGVEESDLIIPKSLAGP LSHHNTREGY RTQVKGPWHS EELEIRFEEC PGTKVHVEET CGTRGPSLRSTTASGRVIEE WCCRECTMPP LSFRAKDGCW YGMEIRPRKE PESNLVRSMV TA,SEQ ID NO: 4 Zika virus NS1 β-ladder domain aa 191-352REAAHSDLGY WIESEKNDTW RLKRAHLIEM KTCEWPKSHT LWTDGVEESD LIIPKSLAGPLSHHNTREGY RTQVKGPWHS EELEIRFEEC PGTKVHVEET CGTRGPSLRS TTASGRVIEEWCCRECTMPP LSFRAKDGCW YGMEIRPRKE PESNLVRSMV TA,SEQ ID NO: 5 Tick-borne encephalitis (FSME) virus NS1 wing domainaa 30-180 according to UniProt ID P14336; European subtype strainNeudoerfl; X = A or C or SDNYAYYPETP GALASAIKET FEEGSCGVVP QNRLEMAMWR SSVTELNLAL AEGEANLTVVVDKFDPTDYR GGVPGLLKKG KDIKVSWKSW GHSMIWSIPE APRRFMVGTE GQSECPLERRKTGVFTVAEF GVGLRTKVFL DFRQEPTHE X  D,SEQ ID NO: 6: Tick-borne encephalitis (FSME) virus NS1 full length aa777-1128 according to UniProt ID P14336; European subtype strainNeudoerfl; 352 amino acidsDVGCAVDTER MELRCGEGLV VWREVSEWYD NYAYYPETPG ALASAIKETF EEGSCGVVPQNRLEMAMWRS SVTELNLALA EGEANLTVVV DKFDPTDYRG GVPGLLKKGK DIKVSWKSWGHSMIWSIPEA PRRFMVGTEG QSECPLERRK TGVFIVAEFG VGLRTKVFLD FRQEPTHECDTGVMGAAVKN GMAIHTDQSL WMRSMKNDTG TYIVELLVTD LRNCSWPASH TIDNADVVDSELFLPASLAG PRSWYNRIPG YSEQVKGPWK YTPIRVIREE CPGTTVTINA KCDKRGASVRSTTESGKVIP EWCCRACTMP PVTFRTGTDC WYAMEIRPVH DQGGLVRSMV VASEQ ID NO: 7 Dengue virus type 1 NS1 wing domain aa 30-180;  X =A or C or SEQYKFQADSP KRLSAAIGKA WEEGVCGIRS ATRLENIMWK QISNELNHIL LENDMKFTVV VGDVAGILAQGKKMIRPQPM EHKYSWKSWG KAKIIGADVQ NTTFIIDGPN TPECPDDQRA WNIWEVEDYG FGIFTTNIWLKLRDSYTQV X  D,SEQ ID NO: 8 Dengue virus type 1 NS1 full length aa 776-1127 accordingto UniProt ID W8FUV0, 352 amino acidsDSGCVINWKG RELKCGSGIF VTNEVHTWTE QYKFQADSPK RLSAAIGKAW EEGVCGIRSATRLENIMWKQ ISNELNHILL ENGMKFTVVV GEVNGILAQG KKMIRPQPME HKYSWKSWGKAKVIGADVQN TTFIIDGPNT PECPDDQRAW NIWEVEDYGF GIFTTNIWLK LRDSYTQVCDHRLMSAAIKD SKAVHADMGY WIESEKNETW KLARASFIEV KTCIWPKSHT LWSNGVLESEMIIPKIYGGP ISQHNYRPGY FTQTAGPWHL GKLELDFELC EGTTVVVDEH CGNRGPSLRTTTVTGKIIHE WCCRSCTLPP LRFKGEDGCW YGMEIRPVKE KEENLVKSMV SA,SEQ ID NO: 9 Dengue virus type 2 NS1 wing domain aa 30-180; X =A or C or SEQYKFQPESP SKLASAIQKA HEEGICGIRS VTRLENLMWK QITPELNHIL SENEVKLTIM TGDIKGIMQAGKRSLRPQPT ELKYSWKTWG KAKMLSTESH NQTFLIDGPE TAECPNTNRA WNSLEVEDYG FGVFTTNIWLKLKEKQDVF X  D,SEQ ID NO: 10 Dengue virus type 2 NS1 full length aa 776-1127 accordingto UniProt ID P29990, strain Thailand/16881/1984, 352 amino acidsDSGCVVSWKN KELKCGSGIF ITDNVHTWTE QYKFQPESPS KLASAIQKAH EEGICGIRSVTRLENLMWKQ ITPELNHILS ENEVKLTIMT GDIKGIMQAG KRSLRPQPTE LKYSWKTWGKAKMLSTESHN QTFLIDGPET AECPNTNRAW NSLEVEDYGF GVFTTNIWLK LKEKQDVFCDSKLMSAAIKD NRAVHADMGY WIESALNDTW KIEKASFIEV KNCHWPKSHT LWSNGVLESEMIIPKNLAGP VSQHNYRPGY HTQITGPWHL GKLEMDFDFC DGTTVVVTED CGNRGPSLRTTTASGKLITE WCCRSCTLPP LRYRGEDGCW YGMEIRPLKE KEENLVNSLV TA,SEQ ID NO: 11 Dengue virus type 3 NS1 wing domain aa 30-180; X =A or C or S)EQYKFQADSP KRLATAIAGA WENGVCGIRS TTRMENLLWK QIANELNYIL WENNIKLTVV VGDIIGILEQGKRTLTPQPM ELKYSWKTWG KAKIVTAETQ NSSFIIDGPN TPECPNASRA WNVWEVEDYG FGVFTTNIWLKLREMYSQL X  D,SEQ ID NO: 12 Dengue virus type 3 NS1 full length aa 774-1125 accordingto UniProt ID W8FRG8, 352 amino acidsDMGCVINWKG KELKCGSGIF VTNEVHTWTE QYKFQADSPK RLATAIAGAW ENGVCGIRSTTRMENLLWRQ IANELNYILW ENNIKLTVVV GDIIGILEQG KRTLTPQPME LKYSWKTWGKAKIVTAETQN SSFIIDGPNT PECPNASRAW NVWEVEDYGF GVFTTNIWLK LREMYSQLCDHRLMSAAVKD ERAVHADMGY WIESQKNGSW KLEKASLIEV KTCTWPKSHT LWSNGVLESDMIIPKSLAGP ISQHNYRPGY HTQTAGPWHL GKLELDFNYC EGTTVVITEN CGTRGPSLRTTTVSGKLIHE WCCRSCTLPP LRYMGEDGCW YGMEIRPINE KEENMVKSLV SA,SEQ ID NO: 13 Dengue virus type 4 NS1 wing domain aa 30-180; X =A or C or S)EQYKFQPESP ARLASAILNA HKDGVCGIRS TTRLENIMWK QITNELNYVL WEGGHDLTVV AGDVKGVLTKGKRALTPPVN DLKYSWKTWG KAKIFTPEAR NSTFLIDGPD TSECPNERRA WNFFEVEDYG FGMFTTNIWMKFREGSSEV X  D,SEQ ID NO: 14 Dengue virus type 4 NS1 full length aa 775-1126 accordingto UniProt ID Q58HT7, strain Philippines/H241/1956, 352 amino acidsDTGCAVSWSG KELKCGSGIF VIDNVHTWTE QYKFQPESPA RLASAILNAH EDGVCGIRSTTRLENIMWKQ ITNELNYVLW EGGHDLIVVA GDVKGVLSKG KRALAPPVND LKYSWKTWGKAKIFTPEAKN STFLIDGPDT SECPNERRAW NFLEVEDYGF GMFTTNIWMK FREGSSEVCDHRLMSAAIKD QKAVHADMGY WIESSKNQTW QIEKASLIEV KTCLWPKTHT LWSNGVLESQMLIPKAYAGP FSQHNYRQGY ATQTVGPWHL GKLEIDFGEC PGTTVTIQED CDHRGPSLRTTTASGKLVTQ WCCRSCTMPP LRFLGEDGCW YGMEIRPLSE KEENMVKSQV SA,SEQ ID NO: 15 West-Nile virus NS1 wing domain aa 30-180; X = A or C or SDRYKFYPETP QGLAKIIQKA HAEGVCGLRS VSRLEHQMWE AIKDELNTLL KENGVDLSVV VEKQNGMYKAAPKRLAATTE KLEMGWKAWG KSIIFAPELA NNTFVIDGPE TEECPTANRA WNSMEVEDFG FGLTSTRMFLRIRETNTTE X  D,SEQ ID NO: 16 West-Nile virus NS1 full length aa 788-1139 accordingto UniProt ID P06935, 352 amino acidsDTGCAIDIGR QELRCGSGVF IHNDVEAWMD RYKFYPETPQ GLAKIIQKAH AEGVCGLRSVSRLEHQMWEA IKDELNTLLK ENGVDLSVVV EKQNGMYKAA PKRLAATTEK LEMGWKAWGKSIIFAPELAN NTFVIDGPET EECPTANRAW NSMEVEDFGF GLTSTRMFLR IRETNTTECDSKIIGTAVKN NMAVHSDLSY WIESGLNDTW KLERAVLGEV KSCTWPETHT LWGDGVLESDLIIPITLAGP RSNHNRRPGY KTQNQGPWDE GRVEIDFDYC PGTTVTISDS CEHRGPAARTTTESGKLITD WCCRSCTLPP LRFQTENGCW YGMEIRPTRH DEKTLVQSRV NA,SEQ ID NO: 17 Yellow fever virus NS1 wing domain aa 30-180; X  =A or C or SNKYSYYPEDP VKLASIVKAS FEEGKCGLNS VDSLEHEMWR SRADEINAIF EENEVDISVVVQDPKNVYQR GTHPFSRIRD GLQYGWKTWG KNLVFSPGRK NGSFIIDGKS RKECPFSNRVWNSFQIEEFG TGVFTTRVYM DAVFEYTID X  D,SEQ ID NO: 18 Yellow fever virus NS1 full length aa 779-1130 accordingto UniProt ID P03314, strain 17D vaccine, 352 amino acidsDQGCAINFGK RELKCGDGIF IFRDSDDWLN KYSYYPEDPV KLASIVKASF EEGKCGLNSVDSLEHEMWRS RADEINAIFE ENEVDISVVV QDPKNVYQRG THPFSRIRDG LQYGWKTWGKNLVFSPGRKN GSFIIDGKSR KECPFSNRVW NSFQIEEFGT GVFTTRVYMD AVFEYTIDCDGSILGAAVNG KKSAHGSPTF WMGSHEVNGT WMIHTLEALD YKECEWPLTH TIGTSVEESEMFMPRSIGGP VSSHNHIPGY KVQTNGPWMQ VPLEVKREAC PGTSVIIDGN CDGRGKSTRSTTDSGKVIPE WCCRSCTMPP VSFHGSDGCW YPMEIRPRKT HESHLVRSWV TA,SEQ ID NO: 19 Japanese encephalitis virus NS1 wing domain aa 30-180; X =A or C or SDRYKYLPETP RSLAKIVHKA HKEGVCGVRS VTRLEHQMWE AVRDELNVLL KENAVDLSVVVNKPVGRYRS APKRLSMTQE KFEMGWKAWG KSILFAPELA NSTFVVDGPE TKECPDEHRAWNSMQIEDFG FGITSTRVWL KIREESTDE X  D,SEQ ID NO: 20 Japanese encephalitis virus NS1 full length aa 795-1146according to UniProt ID Q9YJ16, 352 amino acidsDTGCAIDITR KEMRCGSGIF VHNDVEAWVD RYKYLPETPR SLAKIVHKAH KEGVCGVRSVTRLEHQMWEA VRDELNVLLK ENAVDLSVVV NKPVGRYRSA PKRLSMTQEK FEMGWKAWGKSILFAPELAN STFVVDGPET KECPDEHRAW NSMQIEDFGF GITSTRVWLK IREESTDECDGAIIGTAVKG HVAVHSDLSY WIESRYNDTW KLERAVFGEV KSCTWPETHT LWGDGVEESELIIPHTIAGP KSKHNRREGY KTQNQGPWDE NGIVLDFDYC PGTKVTITED CGKRGPSVRTTTDSGKLITD WCCRSCSLPP LRFRTENGCW YGMEIRPVRH DEATLVRSQV DA,SEQ ID NO: 21 Fusion protein of tandem E. coli SlyD with Zika virusNS1 wing domain aa 30-180MKVAKDLVVS LAYQVRTEDG VLVDESPVSA PLDYLHGHGS LISGLETALE GHEVGDKFDVAVGANDAYGQ YDENLVQRVP KDVFMGVDEL QVGMRFLAET DQGPVPVEIT AVEDDHVVVDGNHMLAGQNL KFNVEVVAIR EATEEELAHG HVHGAHDHHH DHDHDGGGSG GGSGGGSGGGSGGGSGGGKV AKDLVVSLAY QVRTEDGVLV DESPVSAPLD YLHGHGSLIS GLETALEGHEVGDKFDVAVG ANDAYGQYDE NLVQRVPKDV FMGVDELQVG MRFLAETDQG PVPVEITAVEDDHVVVDGNH MLAGQNLKFN VEVVAIREAT EEELAHGHVH GAHDHHHDHD HDGGGSGGGSGGGSGGGSGG GSGGGDRYKY HPDSPRRLAA AVKQAWEEGI CGISSVSRME NIMWKSVEGELNAILEENGV QLTVVVGSVK NPMWRGPQRL PVPVNELPHG WKAWGKSYFV RAAKTNNSFVVDGDTLKECP LKHRAWNSFL VEDHGFGVFH TSVWLKVRED YSLEADLEHH HHHH,SEQ ID NO: 22 Fusion protein of tandem E. coli SlyD with Zika virusNS1 β-ladder domain (aa 191-352, strain Mr 766)MKVAKDLVVS LAYQVRTEDG VLVDESPVSA PLDYLHGHGS LISGLETALE GHEVGDKFDVAVGANDAYGQ YDENLVQRVP KDVFMGVDEL QVGMRFLAET DQGPVPVEIT AVEDDHVVVDGNHMLAGQNL KFNVEVVAIR EATEEELAHG HVHGAHDHHH DHDHDGGGSG GGSGGGSGGGSGGGSGGGKV AKDLVVSLAY QVRTEDGVLV DESPVSAPLD YLHGHGSLIS GLETALEGHEVGDKFDVAVG ANDAYGQYDE NLVQRVPKDV FMGVDELQVG MRFLAETDQG PVPVEITAVEDDHVVVDGNH MLAGQNLKFN VEVVAIREAT EEELAHGHVH GAHDHHHDHD HDGGGSGGGSGGGSGGGSGG GSGGGREAAH SDLGYWIESE KNDTWRLKRA HLIEMKTAEW PKSHTLWTDGVEESDLIIPK SLAGPLSHHN TREGYRTQVK GPWHSEELEI RFEECPGTKV YVEETCGTRGPSLRSTTASG RVIEEWCCRE CTMPPLSFRA KDGCWYGMEI RPRKEPESNL VRSMVTALEH HHHHH,SEQ ID NO: 23 Fusion protein of E. coli SlyD with Zika virus NS1β-ladder domain (aa 191-352, strain Mr 766)MKVAKDLVVS LAYQVRTEDG VLVDESPVSA PLDYLHGHGS LISGLETALE GHEVGDKFDVAVGANDAYGQ YDENLVQRVP KDVFMGVDEL QVGMRFLAET DQGPVPVEIT AVEDDHVVVDGNHMLAGQNL KFNVEVVAIR EATEEELAHG HVHGAHDHHH DHDHDGGGSG GGSGGGSGGGSGGGSGGGRE AAHSDLGYWI ESEKNDTWRL KRAHLIEMKT AEWPKSHTLW TDGVEESDLIIPKSLAGPLS HHNTREGYRT QVKGPWHSEE LEIRFEECPG TKVYVEETCG TRGPSLRSTTASGRVIEEWC CRECTMPPLS FRAKDGCWYG MEIRPRKEPE SNLVRSMVTA LEHHHHHH,

DETAILED DESCRIPTION OF THE INVENTION

Currently available ELISA format immunoassays for detecting anti-Zikavirus antibodies employ an NS1 antigen as immunoreactive reagent.However, we find that these assays lack specificity, leading to a fairlyhigh number of false positive results. Surprisingly, by confining theZika NS1 antigen to its wing domain as explained further below, thenumber of erroneously reactive samples can be considerably reduced whilethe high sensitivity of the assay is maintained.

When samples positive for anti-Zika virus antibodies were tested withtwo different fragments of the NS1 antigen of Zika, i.e. with theso-called “wing” domain antigen and the so-called “ß-ladder” domainantigen it became evident that both antigens are able to detectanti-Zika antibodies. However, we found out that the wing domain antigendoes not cross-react with Dengue antibody positive samples whereas theZika NS1 ß-ladder domain antigen does cross-react with Dengue antibodypositive samples, leading to false positive results and to erroneousconclusions. Additional blocking experiments with anti-Zika positivesera and NS1 antigens of related arboviruses conclusively show that theZika NS1 wing domain antigen signal can hardly be quenched by theserelated arboviral NS1 antigens whereas the Zika NS1 ß-ladder domainantigen signal is quenched significantly. We infer that the ß-ladderantigen is considerably blocked from binding to the immunoglobulins bythe competing related arboviral NS1 antigens. Thus, we were able to showthat the Zika NS1 wing domain antigen is less susceptible toimmunological cross-reactivity with other arboviral NS1 homologues. As aconsequence, the Zika NS1 wing domain enables an immunoassay foranti-Zika antibodies with superior specificity that is capable ofdiagnosing Zika virus infections in the presence of other (recent orpast) arbovirus infections, in an embodiment discriminating Zika virusinfections from Dengue virus infections.

The invention therefore concerns a polypeptide suitable for detectingantibodies against Zika virus in an isolated biological samplecomprising a Zika antigen with a Zika virus NS1 wing domain specificamino acid sequence wherein no amino acid sequences from the Zika virusNS1 ß-ladder domain are present in said polypeptide. In an embodiment,no further Zika virus NS1 specific amino acid sequences are present inthis polypeptide, in an embodiment no further Zika virus specific aminoacid sequences are present in said polypeptide. In an embodiment, saidZika virus NS1 domain specific amino acid sequence consists essentiallyof SEQ ID NOs. 1 or 2, in an embodiment consists of SEQ ID NOs 1 or 2.In particular, no amino acid sequences from the C-terminal so-calledß-ladder domain are present in the polypeptide of the invention. In anembodiment SEQ ID NO:4 is not present in said polypeptide.

In an embodiment, the invention concerns the use of said polypeptide inan immunoassay method for detecting anti-Zika virus antibodies.

The term “Zika virus” is equivalent to the shorter forms “Zika” or“ZIKV”; these acronyms refer to the same virus.

The terms “NS1”, “NS1 antigen”, NS1 polypeptide” are used synonymouslyand refer to the non-structural antigen no. 1 within the viral precursorpolyprotein and relate (unless specified differently) to the full lengthantigen NS1. The structure of this protein has been described for WestNile virus and Dengue virus 2 in Akey et al, supra. For Zika NS1 thestructure of the C-terminal domain (amino acid residues 172-352) hasbeen described by Song et al. (supra) and the complete NS1three-dimensional structure has been described in further detail byBrown et al. (supra). The Zika NS1 sequence comprises 352 amino acidsand is shown in SEQ ID NO: 3. The term “NS1 wing” or “NS1 wing domain”,“variant of NS1 wing” or “NS1 wing region” refers to a domain within theNS1 polypeptide and is therefore a partial sequence of NS1. For the ZikaNS1 wing domain this is exemplified in SEQ ID NOs:1 and 2. Also theterms “polypeptide”, “polypeptides”, “antigen” and “antigens” areunderstood as synonyms unless further specified.

The synonymous terms “ß-ladder”, “ß-ladder domain” or “ladder tipantigen” or “ladder tip”, “ladder tip domain”, “ladder tip polypeptide”,“ladder tip antigen” refer to a NS1 domain located C-terminally adjacentto the wing domain. This domain has been described for West Nile virusand Dengue virus 2 in Akey et al, supra. For Zika virus this NS1 domainhas been described by Song et al., supra, and Brown et al., supra. Forthe Zika NS1 ß-ladder domain the amino acid sequence is exemplified inSEQ ID NO:4.

These definitions are applicable to all arboviruses within thisspecification. The following arboviruses that belong to the flavivirusfamily can be abbreviated as follows: West Nile virus (West Nile, WNV),tick-borne encephalitis virus (TBEV or FSME), Dengue virus 1-4 (Dengue,the four virus strains of Dengue: DENV 1-4), yellow fever virus (YFV),Japanese encephalitis virus (JEV).

According to the invention, a Zika virus NS1 domain specific amino acidsequence is an amino acid sequence wherein no amino acid sequences fromthe Zika virus NS1 ß-ladder domain are present in said polypeptide. Inan embodiment, no further Zika virus specific amino acid sequences arepresent in such polypeptide. For example a Zika NS1 wing domainpolypeptide contains only a wing domain sequence, in an embodimentcontains SEQ ID NOs. 1 or 2. In an embodiment, SEQ ID NO:4 is notpresent in said polypeptide sequence. In another embodiment, no furtherZika virus specific amino acid sequences are present in this sequence.The absence of NS1 ß-ladder domain specific sequences and in anembodiment the absence of further Zika NS1 specific sequences or offurther Zika specific sequences supports the aim to either reduce orcompletely avoid cross-reactivities with antibodies raised against otherarboviruses.

However, variants of the Zika NS1 wing domain polypeptide areencompassed as well. These variants may easily be created by a personskilled in the art by conservative or homologous substitutions of thedisclosed amino acid sequences (such as e.g. substitutions of a cysteineby alanine or serine, or substitutions of isoleucine by valine, or viceversa). The term “variants” in this context also relates to a protein ora protein fragment (i.e. a polypeptide or peptide) substantially similarto said protein. For example, modifications such as C- or N-terminaltruncations at one end or at both ends by 1 to 10 amino acids, in anembodiment by 1 to 5 amino acids, are within the scope of the claimedZika NS1 wing domain antigens. In particular, a variant may be anisoform which shows amino acid exchanges, deletions or insertionscompared to the amino acid sequence of the most prevalent proteinisoform. In one embodiment, such a substantially similar protein has asequence similarity to the most prevalent isoform of the protein of atleast 80%, in another embodiment at least 85% or at least 90%, in yetanother embodiment at least 95%. The term “variant” also relates to apost-translationally modified protein such as a glycosylated orphosphorylated protein. According to the invention a variant classifiesas a Zika NS1 wing domain variant as long as the immunoreactivity in anin vitro diagnostic immunoassay is unchanged or largely maintained, i.e.the variant is still able to bind and detect anti-Zika antibodiespresent in an isolated sample while antibodies raised against otherarboviruses are not detected or detected to a much lower extent. Inaddition, the overall three-dimensional structure of said Zikapolypeptide remains unaltered, so that epitopes that were previously(i.e. in the wild type) present and accessible for binding to antibodiesare still present and accessible in the variant.

A “variant” is also a protein or antigen which has been modified forexample by covalent or non-covalent attachment of a label or carriermoiety to the protein or antigen. Possible labels are radioactive,fluorescent, chemiluminescent, electrochemiluminescent, enzymes orothers e.g. like digoxin, digoxigenin or biotin. These labels are knownto a person skilled in the art.

When a provided polypeptide sequence information specified in the formof SEQ ID NOs is described by the term “consisting essentially of”(i.e., said sequence) this means that the sequence is present asliterally listed but can also be present as variants that do notmaterially affect the basic characteristics of this polypeptide in termsof immunological binding to antibodies. An example of this would be thedeletion or addition of only few amino acids at the N- and/or C-terminalend of this peptide as well as the exchange of a similar amino acid as,e.g., alanine for serine, isoleucine for valine, and vice versa.

The Zika NS1 wing domain antigens of the current invention are soluble,stable and immunoreactive, i.e. they are suitable as antigens for use inan immunological assay. This means that the antigens according to theinvention are soluble under physiological buffer conditions, for examplein a phosphate buffer system at ambient temperature without addition ofdetergents. The antigens are also capable of binding to or beingrecognized and bound by antibodies specific for Zika NS1 wing domain,like e.g. anti-Zika antibodies present in an isolated sample such ashuman sera.

In an embodiment, the addition of non-Zika-specific linker or peptidicfusion amino acid sequences to the Zika NS1 wing domain polypeptides ispossible as these sequences are not specific for anti-Zika virusantibodies and would not interfere with the in vitro diagnosticimmunoassay.

In an embodiment the Zika NS1 wing domain antigens may be fused to achaperone. The term “fusion protein”, “fusion polypeptide” or “fusionantigen” refers to a protein comprising a Zika NS1 wing domainpolypeptide and at least one protein part derived from a chaperone thatserves the role of a fusion partner.

Chaperones are well-known folding helper proteins that assist thefolding and maintenance of the structural integrity of other proteins.Examples of folding helpers are described in detail in WO 03/000877.According to the invention chaperones of the peptidyl prolyl isomeraseclass such as chaperones of the FKBP family can be used for fusion tothe Zika NS1 wing domain antigen variants. Examples of FKBP chaperonessuitable as fusion partners are FkpA, SlyD and SlpA. A further chaperonesuitable as a fusion partner for the Zika NS1 wing antigen is Skp, atrimeric chaperone from the periplasm of E. coli, not belonging to theFKBP family. It is not always necessary to use the complete sequence ofa chaperone. Functional fragments of chaperones (so-calledbinding-competent modules or polypeptide-binding motifs) which stillpossess the required abilities and functions may also be used (cf. WO98/13496).

In a further embodiment of the invention at least one or at least twomodules of an FKBP chaperone such as e.g. E. coli SlyD, SlpA or FkpA areused as fusion moieties for expression of the Zika NS1 wing domainantigen. The chaperone Skp may be used as a fusion partner as well. Thefusion of two FKBP-chaperone domains results in improved solubility ofthe resulting fusion polypeptide. The fusion moieties may be located atthe N-terminus or at the C-terminus or at both ends (sandwich-like) ofthe Zika NS1 wing domain antigen.

In an embodiment the Zika NS1 wing domain antigen is fused to anoligomeric chaperone. Oligomeric chaperones are chaperones thatnaturally form dimers, trimers or even higher multimers so that aplurality of monomeric subunits are assembled into a well-definedfunctional quaternary structure by specific non-covalent interactions.Thereby, the covalently fused antigens are coerced into a higher epitopedensity as well. Preferred oligomeric chaperones are FkpA and Skp.Multimerized antigens are particularly useful in detecting IgMantibodies and hence early immune responses immediately afterinfections.

In an embodiment, the Zika NS1 wing domain polypeptide is fused to one,two or more chaperone molecules of bacterial SlyD, SlpA, FkpA or Skp, inan embodiment of E. coli SlyD, SlpA, FkpA or Skp. In a furtherembodiment the Zika NS1 wing domain polypeptide consists of SEQ IDNO:21.

Another embodiment of the invention is a Zika NS1 antigen that does notimmunologically cross-react with antibodies raised against structurallyrelated antigens from tick-borne encephalitis virus comprising any ofSEQ ID NOs:5 or 6, and/or from Dengue virus 1-4 comprising any of SEQ IDNOs:7 to 14, and/or from West Nile virus comprising any of SEQ ID NOs:15or 16, and/or from yellow fever virus comprising any of SEQ ID NOs:17 or18, and/or from Japanese encephalitis virus comprising any of SEQ IDNOs:19 to 20, but immunologically reacts with antibodies raised againstfull length Zika virus NS1 antigen according to SEQ ID NO:3. In afurther embodiment said Zika NS1 antigen is a Zika NS1 wing domainantigen wherein the Zika-specific sequence consists essentially of SEQID NO: 1 or 2, in an embodiment consists of SEQ ID NO: 1 or 2.

The term “does not immunologically cross-react” designates a stronglyreduced or completely abolished undesired immunological reactivity. Theterm “immunological cross-reactivity” has been coined to illustrate anunwanted binding of immunoglobulins which is due to similarities insequence or structure of an antigen with the immunogen, against whichthe antibodies have originally been mounted. In an embodiment, Zikavirus NS1 wing domain polypeptides show a completely abolished orstrongly reduced immunological reactivity towards antibodies or towardsa subset of antibodies raised against homologous or related thecorresponding arboviral NS1 antigens named above as compared to the fulllength Zika virus NS1 polypeptide. In yet another embodiment Zika virusNS1 wing domain polypeptides show a strongly reduced immunologicalcross-reactivity towards antibodies or towards a subset of antibodiesraised against Dengue virus, in an embodiment towards antibodies raisedagainst Dengue virus types 1, 2, 3, 4. In a further embodiment, astrongly reduced immunological cross-reactivity of the Zika virus NS1wing domain polypeptides applies also to antibodies or a subset ofantibodies raised against yellow fever virus.

The expression “does not immunologically cross-react” also refers to asituation where in an immunoassay in a double antigen sandwich formatfor detecting antibodies the sample antibodies (i.e., the analyteantibodies) are bound by two specific antigens: one is capable of beingbound to a solid phase and the other carries a label, the sampleantibody is sandwiched between both antigens. In the presence of analyteantibodies the labeled antigen is recruited—within the resulting ternaryimmune complex—to the solid phase and yields a signal. In the presentcase, a Zika NS1 wing domain polypeptide is labeled and the measuredsignal is set as 100%. In a parallel or subsequent experiment, the sameassay is run with another aliquot of the same (positive) sample and inaddition a non-labeled antigen with an amino acid sequence suspected tocompete with the labeled antigen is added to the mixture. In the presentcase, full length NS1 polypeptides of either TBEV, DENV1-4, WNV, YFV orJEV are added. In another embodiment, NS1 polypeptides consisting of orcomprising only the NS1 wing domain of either TBEV, DENV1-4, WNV, YFV orJEV are added. When the signal obtained after measurement is maintainedat about at least 70% signal recovery, in an embodiment at least 80%signal recovery, in an embodiment at least 85% signal recovery, in anembodiment at least 90% signal recovery of the original signal, theflaviviral (in this example: Zika) NS1 polypeptide is not prone tosignal quenching. It is not outcompeted by the added antigens andtherefore withstands potentially cross-reactive substances. Forillustration, such blocking experiments are described in example 2(table 2).

In an embodiment, NS1 full length or NS1 wing domain peptides fromtick-borne encephalitis virus comprising any of SEQ ID NOs:5 or 6,and/or from Dengue virus 1-4 comprising any of SEQ ID NOs:7 to 14,and/or from West Nile virus comprising any of SEQ ID NOs:15 or 16,and/or from yellow fever virus comprising any of SEQ ID NOs:17 or 18,and/or from Japanese encephalitis virus comprising any of SEQ ID NOs:19to 20, are added in the above-described blocking experiment.

In another embodiment, the Zika NS1 wing domain polypeptideimmunologically reacts with antibodies, or a subset of antibodies,raised against full-length NS1 antigen as shown in SEQ ID NO:3. Thismeans that in the above-disclosed assay setup, the signal of the ZikaNS1 wing domain polypeptide should be fully quenched (100%) uponaddition of the full-length Zika NS1 polypeptide.

The approach how to determine immunological cross-reactivity toZika-related viruses is further described in example 2.

The Zika NS1 polypeptides (both wing domain and ß-ladder domain) as wellas the polypeptides applied for the blocking experiments of example 2can be generated and prepared by means of recombinant DNA techniques andprotein purification techniques as known in the art. Another aspect ofthe invention therefore is a recombinant DNA molecule encoding a ZikaNS1 wing domain antigen, in an embodiment an antigen according to SEQ IDNOs 1, 2 or 21 and variants thereof as defined further above.

The term “recombinant DNA molecule” refers to a molecule which is madeby the combination of two otherwise separated segments of DNA sequenceaccomplished by the artificial manipulation of isolated segments ofpolynucleotides by genetic engineering techniques or by chemicalsynthesis. In doing so one may join together polynucleotide segments ofdesired functions to generate a desired combination of functions.Recombinant DNA techniques for expression of proteins in prokaryotic orlower or higher eukaryotic host cells are well known in the art. Theyhave been described e.g. by Sambrook et al., (1989, Molecular Cloning: ALaboratory Manual)

The recombinant DNA molecules according to the invention may alsocontain sequences encoding linker peptides of 5 to 100 amino acidresidues in between the Zika virus NS1 wing domain antigen and thefusion moieties and also between several fusion moieties. Such a linkersequence may for example harbor a proteolytic cleavage site.

A further aspect of the invention is an expression vector comprisingoperably linked a recombinant DNA molecule according to the presentinvention, i.e., a recombinant DNA molecule encoding a Zika virus NS1wing domain antigen and optionally a peptidyl prolyl isomerasechaperone, such as an FKBP-chaperone, wherein the FKBP-chaperone isselected from FkpA, SlyD and SlpA. In an alternative embodiment therecombinant DNA molecule encodes a fusion protein comprising a Zikavirus NS1 wing domain antigen and Skp. The expression vector comprisinga recombinant DNA according to the present invention may be used toexpress the Zika virus NS1 wing domain antigen in a cell freetranslation system or may be used to transform a host cell forexpression of the Zika virus NS1 wing domain antigen according tomethods well known in the art. Another aspect of the invention thereforerelates to a host cell transformed with an expression vector accordingto the present invention. In one embodiment of the current invention therecombinant Zika virus NS1 wing domain antigens are produced in E. colicells.

An additional aspect is a method for producing a soluble, stable andimmunoreactive Zika virus NS1 wing domain antigen. Said Zika virus NS1wing domain antigen may be produced as a fusion protein containing theZika virus NS1 wing domain antigen and a chaperone. Preferably, achaperone such as Skp or a peptidyl prolyl isomerase class chaperonelike an FKBP chaperone is used. In a further embodiment of the inventionsaid FKBP chaperone is selected from the group consisting of SlyD, FkpAand SlpA.

This method comprises the steps of

-   a) culturing host cells transformed with the above-described    expression vector containing a gene encoding an Zika virus NS1 wing    domain antigen-   b) expression of the gene encoding said Zika virus NS1 wing domain    antigen-   c) purification of said Zika virus NS1 wing domain antigen.

Optionally, as an additional step d), functional solubilization needs tobe carried out so that the Zika virus NS1 wing domain antigen is broughtinto a soluble and immunoreactive conformation by means of refoldingtechniques known in the art.

Yet another embodiment is a method for producing a soluble, stable andimmunoreactive Zika virus NS1 wing domain antigen in a cell-free invitro translation system.

An additional aspect of the present invention concerns a method for thedetection of anti-Zika antibodies in an isolated human sample wherein aZika virus NS1 wing domain antigen according to the invention is used asa binding partner for the antibodies. The invention thus covers a methodfor the detection of antibodies specific for Zika virus in an isolatedsample, said method comprising

a) forming an immunoreaction admixture by admixing a body fluid samplewith an Zika virus NS1 wing domain antigen according to the inventionb) maintaining said immunoreaction admixture for a time periodsufficient for allowing antibodies against said Zika virus NS1 wingdomain antigen present in the body fluid sample to immunoreact with saidZika virus NS1 wing domain antigen to form an immunoreaction product;and c) detecting the presence and/or the concentration of any of saidimmunoreaction product.

In a further aspect said method is suitable for detecting Zika virusantibodies of the IgG and the IgM subclass or of both classes in thesame immunoassay.

Immunoassays for detection of antibodies are well known in the art, andso are methods for carrying out such assays and practical applicationsand procedures. The Zika NS1 antigens according to the invention can beused to improve assays for the detection of anti-Zika antibodiesindependently of the labels used and independently of the mode ofdetection (e.g., radioisotope assay, enzyme immunoassay,electrochemiluminescence assay, etc.) or the assay principle (e.g., teststrip assay, sandwich assay, indirect test concept or homogenous assay,etc.).

In an embodiment of the invention the immunoassay is a particle-basedimmunoassay applying microparticles as solid phase. A “particle” as usedherein means a small, localized object to which can be ascribed aphysical property such as volume, mass or average size. Microparticlesmay accordingly be of a symmetrical, globular, essentially globular orspherical shape, or be of an irregular, asymmetric shape or form. Thesize of a particle envisaged by the present invention may vary. In oneembodiment the microparticles used are of globular shape, e.g.microparticles with a diameter in the nanometer and micrometer range. Inone embodiment the microparticles used in a method according to thepresent disclosure have a diameter of 50 nanometers to 20 micrometers.In a further embodiment the microparticles have a diameter of between100 nm and 10 μm. In one embodiment, the microparticles used in a methodaccording to the present disclosure have a diameter of 200 nm to 5 μm orfrom 750 nm to 5 μm.

Microparticles as defined herein above may comprise or consist of anysuitable material known to the person skilled in the art, e.g. they maycomprise or consist of or essentially consist of inorganic or organicmaterial. Typically, they may comprise or consist of or essentiallyconsist of metal or an alloy of metals, or an organic material, orcomprise or consist of or essentially consist of carbohydrate elements.Examples of envisaged material for microparticles include agarose,polystyrene, latex, polyvinyl alcohol, silica and ferromagnetic metals,alloys or composition materials. In one embodiment the microparticlesare magnetic or ferromagnetic metals, alloys or compositions. In furtherembodiments, the material may have specific properties and e.g. behydrophobic, or hydrophilic. Such microparticles typically are dispersedin aqueous solutions and retain a small negative surface charge keepingthe microparticles separated and avoiding non-specific clustering.

In one embodiment of the present invention, the microparticles areparamagnetic microparticles and the separation of such particles in themeasurement method according to the present disclosure is facilitated bymagnetic forces. Magnetic forces are applied to pull the paramagnetic ormagnetic particles out of the solution/suspension and to retain them asdesired while liquid of the solution/suspension can be removed and theparticles can e.g. be washed.

All biological liquids known to the expert can be used as isolatedsamples for the detection of anti-Zika antibodies. The samples usuallyused are bodily liquids like whole blood, blood serum, blood plasma,urine or saliva, in an embodiment blood serum or plasma.

A further embodiment of the invention is an immunoassay for detectinganti-Zika antibodies in an isolated sample performed according to theso-called double antigen sandwich concept (DAGS). Sometimes this assayconcept is also termed double antigen bridge concept, because the twoantigens are bridged by an antibody analyte. In such an assay theability of an antibody to bind at least two different molecules of agiven antigen with its two (IgG, IgE), four (IgA) or ten (IgM) paratopesis required and utilized.

In more detail, an immunoassay for the determination of anti-Zikaantibodies according to the double antigen bridge format is carried outby incubating a sample containing the anti-Zika antibodies with twodifferent Zika virus NS1 wing domain antigens, i.e. a first (“solidphase” or “capture”) Zika virus NS1 wing domain antigen and a secondZika virus NS1 wing domain (“detection” or “reporter”) antigen, whereineach of the said antigens binds specifically to said anti-Zikaantibodies. The first antigen can be bound directly or indirectly to asolid phase and usually carries an effector group which is part of abioaffine binding pair.

One type of a bioaffine binding pair which is suitable for the methodaccording to the present invention is a hapten and anti-hapten antibodybinding pair. A hapten is an organic molecule with a molecular weight of100 to 2000 Dalton, preferably of 150 to 1000 Dalton. Such smallmolecule can be rendered immunogenic by coupling it to a carriermolecule and anti-hapten antibodies can be generated according tostandard procedures. The hapten may be selected from the groupcomprising sterols, bile acids, sexual hormones, corticoids,cardenolides, cardenolide-glycosides, bufadienolides,steroid-sapogenines and steroid alkaloids, cardenolides andcardenolide-glycosides. Representatives of these substance classes aredigoxigenin, digitoxigenin, gitoxigenin, strophanthidin, digoxin,digitoxin, ditoxin, and strophanthin. Another suitable hapten is forexample fluorescein. In an embodiment, a bioaffine binding paircomprises biotin and avidin/streptavidin or digoxin and anti-digoxin.

In yet another embodiment, the first antigen is conjugated to biotin andthe complementary solid phase is coated with either avidin orstreptavidin. The second antigen carries a label that confers specificdetectability to this antigen molecule, either alone or in complex withother molecules. Thus an immunoreaction admixture is formed comprisingthe first antigen, the sample antibody and the second antigen. Thisternary complex consisting of analyte antibody sandwiched in between twoantigen molecules is termed immunocomplex or immunoreaction product. Asolid phase to which the first antigen can be bound is added eitherbefore the addition of the sample to said antigens or after theimmunoreaction admixture is formed. This immunoreaction admixture ismaintained for a time period sufficient for allowing anti-Zikaantibodies against said Zika virus NS1 wing domain antigens in the bodyfluid sample to immunoreact with said Zika virus NS1 wing domainantigens to form an immunoreaction product. Next step is a separationstep wherein the liquid phase is separated from the solid phase.Finally, the presence of any of said immunoreaction product is detectedin the solid or liquid phase or both.

In said DAGS immunoassay the basic structures of the “solid phaseantigen” and the “detection antigen” are essentially the same. It isalso possible to use, in a double antigen bridge assay, similar butdifferent Zika virus NS1 wing domain antigens, which are immunologicallycross-reactive. The essential requirement for performing such assays isthat the relevant epitope or the relevant epitopes are present on bothantigens. According to the invention it is possible to use the same ordifferent fusion moieties for each Zika virus NS1 wing domain antigen(e.g. SlyD fused to Zika virus NS1 wing domain antigen on the solidphase side and, e.g., FkpA fused to Zika virus NS1 wing domain antigenon the detection side) as such variations significantly alleviate theproblem of non-specific binding and thus mitigate the risk offalse-positive results.

A further embodiment is a method for detecting anti-Zika virusantibodies (i.e., immunoglobulins) of the M class (IgM detection). In anembodiment of this method a Zika NS1 wing domain polypeptide asdisclosed further above is applied in such a way that the multivalentIgM antibodies present in a sample specifically bind to the Zika NS1wing domain antigen. In an embodiment, the Zika NS1 wing domain antigenis provided in a multimeric form by either chemically cross-linking theantigen or by fusing the antigen to an oligomerizing molecule such as anoligomeric chaperone, in an embodiment to FkpA or Skp. In anotherembodiment a Zika wing domain antigen is present in multiple form byconnecting individual antigens in series, adjacent to each other. Theseindividual antigen moieties can also be separated by linker moleculesthat are not Zika specific. In a further embodiment the multiple Zikaantigens connected in series can additionally be multimerized by anoligomerization molecule such as an oligomeric chaperone like e.g. FkpAor Skp. In yet another embodiment the Zika NS1 wing domain polypeptideis used in a multimeric form wherein each polypeptide is present atleast in duplicate form, in an embodiment it is present three to tentimes.

In yet another embodiment of the IgM detection method of Zika-antibodiesthe IgM class antibodies present in the sample are bound to a solidphase by a so-called μ-capture component which usually is a bindingpartner or an antibody or antibody fragment that specifically binds tothe Fc part of human IgM molecules, independently of the specificity ofthe IgM molecule. Said μ-capture component carries an effector group(such as biotin) which is part of a bioaffine pair with avidin orstreptavidin. In an embodiment also other bioaffine pairs such as e.g.digoxin and anti-digoxin or further hapten and anti-haptens as describedfurther above can be used. In an embodiment, a solid phase covered withavidin or streptavidin then attracts and binds the μ-capture component.In order to specifically detect the Zika-specific antibodies a Zika NS1wing domain polypeptide as described is used in a labeled form to detectthe anti-Zika antibodies of the IgM class.

Another embodiment is the use of a Zika NS1 wing domain polypeptide asdetailed above in an in vitro diagnostic test, in an embodiment animmunoassay method as defined above, for the detection of anti-Zikavirus antibodies.

As a further embodiment the maximal total duration of the immunoassaymethod for detecting Zika virus antibodies is less than one hour, i.e.less than 60 minutes, in an embodiment less than 30 minutes, in afurther embodiment less than 20 minutes, in an embodiment between 15 and30 minutes, in an embodiment between 15 to 20 minutes. The durationincludes pipetting the sample and the reagents necessary to carry outthe assay as well as incubation time, optional washing steps, thedetection step and also the final output of the result.

An additional subject matter of the invention is a reagent kit for thedetection of antibodies against Zika virus that comprises a Zika NS1wing domain polypeptide disclosed above. In an embodiment a reagent kitcomprises in separate containers or in separated compartments of asingle container unit at least microparticles coated with avidin orstreptavidin, and a Zika NS1 wing domain polypeptide as detailed before.In another embodiment, said microparticles are coated with one partnerof other bioaffine pairs as described further above, such as e.g.digoxin and anti-digoxin, hapten and anti-hapten. In an embodiment, saidZika NS1 wing domain polypeptide is covalently coupled to biotin. In anembodiment, said Zika NS1 wing domain is covalently coupled to thesecond partner of other bioaffine pairs such as e.g. digoxin andanti-digoxin, hapten and anti-hapten. In another embodiment, said ZikaNS1 wing domain polypeptide is covalently coupled to a detectable label,in an embodiment to an electrochemiluminescent complex. In a furtherembodiment also chemiluminescent labels such as e.g. acridinium ester orradioactive or fluorescent compounds or enzymes can be applied as label.In yet another embodiment, said reagent kit comprises in separatecontainers or in separated compartments of a single container unit atleast microparticles coated with avidin or streptavidin, a first ZikaNS1 wing domain polypeptide covalently coupled to biotin and a secondZika NS1 wing domain polypeptide that is covalently coupled to adetectable label, e.g. to an electrochemiluminescent ruthenium complexor an electrochemiluminescent iridium complex.

A further embodiment is a reagent kit for detecting anti-Zika antibodiesof the IgM class, comprising in separate containers or in separatedcompartments of a single container unit at least microparticles coatedwith avidin or streptavidin, and a μ-capture binding partner that iscovalently coupled to biotin. In a further embodiment said IgM detectionreagent kit additionally contains Zika NS1 wing domain polypeptide whichis covalently coupled to a detectable label, in an embodiment to anelectrochemiluminescent complex.

The term single container unit relates to the fact that for manyautomatic analyzers, like the Elecsys® analyzer series from Rochediagnostics, the reagents required to measure a certain analyte areprovided in the form of a “reagent pack”, i.e. as one container unitfitting on the analyzer and containing in different compartments all thekey reagents required for measurement of the analyte of interest.

In addition, the reagent kits defined above contain controls andstandard solutions as well as reagents in one or more solutions with thecommon additives, buffers, salts, detergents and the like as used by theaverage man skilled in the art along with instructions for use.

In yet another aspect, the invention concerns a method for detectingantibodies against a Zika virus in an isolated biological sample that ispresumed to contain antibodies against at least one other non-Zikaflavivirus such as e.g. Dengue virus. In this method, Zika virus NS1polypeptide comprising a complete or partial sequence of the ß-ladderdomain is applied as specific binding partner, i.e. not only the NS1wing domain. In this setup cross-reactivity against other non-Zikaflaviviruses can be expected due to the presence of ß-ladder domainpeptide sequences in the specific binding partner. In order to eliminatethis interference a polypeptide comprising solely the NS1 ß-ladderdomain of said Zika virus is added in an unlabeled form so that thecross-reacting antibodies of non-Zika origin are bound and quenched. Inan embodiment, the ß-ladder domain is added as a quencher, in a furtherembodiment said ß-ladder domain polypeptide consists essentially of SEQID NO:4, in an embodiment consists of SEQ ID NO:4.

The following embodiments are also part of the invention:

-   -   1. A polypeptide suitable for detecting antibodies against Zika        virus in an isolated biological sample comprising a Zika virus        NS1 wing domain specific amino acid sequence, wherein no amino        acid sequences from the Zika virus NS1 ß-ladder domain are        present in said polypeptide    -   2. A polypeptide according to embodiment 1, wherein no further        Zika virus specific amino acid sequences are present in said        polypeptide.    -   3. A polypeptide according to any of embodiments 1 or 2, wherein        said Zika virus NS1 wing domain specific amino acid sequence        consists essentially of SEQ ID NO. 1 or 2    -   4. A polypeptide according to any of embodiments 1 to 3, wherein        said Zika virus NS1 wing domain specific amino acid sequence        consists of SEQ ID NO. 1 or 2.    -   5. A polypeptide according to embodiment 1 to 4, wherein said        Zika virus NS1 wing domain specific amino acid sequence can be        truncated by 1 to 5 amino acids at its N-terminal or C-terminal        end or at both ends.    -   6. A polypeptide according to embodiments 1 to 5, wherein said        Zika virus NS1 wing domain specific amino acid sequence can be        modified by conservative amino acid substitutions so that the        immunoreactivity of said Zika polypeptide remains unchanged or        is largely maintained, in an embodiment so that the        three-dimensional structure of said Zika polypeptide remains        unchanged.    -   7. A polypeptide according to any of embodiments 1 to 6 wherein        said Zika polypeptide is fused to a chaperone.    -   8. A polypeptide according to any of embodiments 1 to 7 wherein        said chaperone is selected from the group consisting of SlyD,        SlpA, FkpA and Skp.    -   9. A polypeptide according to embodiment 8 consisting        essentially of SEQ ID NO:21, in an embodiment consisting of SEQ        ID NO:21.    -   10. A polypeptide according to any of embodiments 1-9, wherein        in an immunoassay for detecting antibodies against Zika virus,        said Zika NS1 polypeptide does not immunologically cross-react        with antibodies raised against structurally related antigens        from tick-borne encephalitis virus comprising any of SEQ ID        NOs:5 or 6, and/or from Dengue virus 1-4 comprising any of SEQ        ID NOs:7 to 14, and/or from West Nile virus comprising any of        SEQ ID NOs:15 or 16, and/or from yellow fever virus comprising        any of SEQ ID NOs:17 or 18, and/or from Japanese encephalitis        virus comprising any of SEQ ID NOs:19 to 20, but immunologically        reacts with antibodies raised against full length Zika virus NS1        polypeptide according to SEQ ID NO:3.    -   11. A method of producing a soluble and immunoreactive Zika        virus NS1 wing domain polypeptide, said method comprising the        steps of        -   a) culturing host cells transformed with an expression            vector comprising operably linked a recombinant DNA molecule            encoding a Zika virus NS1 polypeptide according to any of            embodiments 1 to 10,        -   b) expression of said Zika virus NS1 polypeptide and        -   c) purification of said Zika virus NS1 polypeptide.    -   12. A method for detecting antibodies specific for Zika virus in        an isolated sample wherein a Zika virus polypeptide according to        any of embodiments 1 to 10 or a Zika virus polypeptide obtained        by a method according to embodiment 11 is used as a capture        reagent and/or as a binding partner for said anti-Zika virus        antibodies.    -   13. A method for detecting antibodies specific for Zika virus in        an isolated sample said method comprising        -   a) forming an immunoreaction admixture by admixing a body            fluid sample with a Zika virus polypeptide according to any            of embodiments 1 to 10 or a Zika virus polypeptide obtained            by the method of embodiment 11        -   b) maintaining said immunoreaction admixture for a time            period sufficient for allowing antibodies present in the            body fluid sample against said Zika virus polypeptide to            immunoreact with said Zika virus polypeptide to form an            immunoreaction product; and        -   c) detecting the presence and/or the concentration of any of            said immunoreaction product.    -   14. A method for detecting antibodies specific for Zika virus in        an isolated sample according to any of embodiments 12 or 13,        wherein the detected antibody is an IgG antibody.    -   15. A method for detecting antibodies specific for Zika virus in        an isolated sample according to embodiment 13 or 14 wherein said        immunoreaction is carried out in a double antigen sandwich        format comprising        -   a) adding to said sample a first Zika virus polypeptide            according to any of embodiments 1 to 10 or a polypeptide            produced by a method according to embodiment 11, which can            be bound directly or indirectly to a solid phase and said            first Zika virus polypeptide carries an effector group which            is part of a bioaffine binding pair, and a second Zika virus            polypeptide according to any of embodiments 1 to 10 or            produced by a method according to embodiment 11, and said            second Zika virus polypeptide carries a detectable label,            wherein said first and second Zika virus polypeptides bind            specifically to said anti-Zika virus antibodies,        -   b) forming an immunoreaction admixture comprising said first            Zika virus polypeptide, said sample antibody and said second            Zika virus polypeptide wherein a solid phase carrying a            corresponding effector group of said bioaffine binding pair            is added before, during or after forming the immunoreaction            admixture,        -   c) maintaining said immunoreaction admixture for a time            period sufficient for allowing Zika virus antibodies against            said first and second Zika virus polypeptides in the body            fluid sample to immunoreact with said first and second Zika            virus polypeptides to form an immunoreaction product,        -   d) separating the liquid phase from the solid phase        -   e) detecting the presence of any of said immunoreaction            product in the solid or liquid phase or both.    -   16. A method for detecting antibodies specific for Zika virus        according to embodiment 15, wherein said first Zika virus        polypeptide carries a biotin moiety, and said second Zika virus        polypeptide is labeled with an electrochemiluminescent moiety,        in an embodiment with a ruthenium or iridium complex.    -   17. A method for detecting antibodies specific for Zika virus in        an isolated sample according to any of embodiments 12 or 13,        wherein the detected antibody is an IgM antibody.    -   18. A method for detecting antibodies specific for Zika virus of        the IgM class according to embodiment 17, wherein the Zika NS1        wing domain polypeptide is used in a multimeric form, in an        embodiment wherein said polypeptide is present at least in        duplicate form, in an embodiment three to ten times.    -   19. A method according to any of embodiments 12 or 13, wherein        said detected antibodies are IgM antibodies and wherein said IgM        antibodies are captured on a solid phase by a μ-capture binding        partner.    -   20. A method for detecting IgM antibodies specific for Zika        virus in an isolated sample according to any of embodiments 17        to 19 wherein said immunoreaction is carried out in a μ-capture        format comprising        -   a) adding to said sample a μ-capture binding partner which            can be bound directly or indirectly to a solid phase and            said μ-capture binding partner carries an effector group            which is part of a bioaffine binding pair, and a Zika virus            polypeptide according to any of embodiments 1 to 10 or            produced by a method according to embodiment 11, and said            Zika virus polypeptide carries a detectable label,        -   wherein said μ-capture binding partner specifically binds to            the Fc part of human IgM antibodies and said Zika virus            polypeptide binds specifically to said anti-Zika virus            antibodies,        -   b) forming an immunoreaction admixture comprising said            μ-capture binding partner, said sample antibodies and said            Zika virus polypeptide wherein a solid phase carrying a            corresponding effector group of said bioaffine binding pair            is added before, during or after forming the immunoreaction            admixture,        -   c) maintaining said immunoreaction admixture for a time            period sufficient for allowing IgM antibodies against said            Zika virus polypeptide in the body fluid sample to            immunoreact with said Zika virus polypeptide to form an            immunoreaction product,        -   d) separating the liquid phase from the solid phase        -   e) detecting the presence of any of said immunoreaction            product in the solid or liquid phase or both.    -   21. A method according to any of embodiments 12 to 20, wherein        said method does not use Zika NS1 antigens from the ß-ladder        domain, in an embodiment does not use a polypeptide comprising        an amino acid sequence according to SEQ ID NO:4.    -   22. Use of a Zika virus polypeptide according to any of        embodiments 1 to 10 or of a Zika virus polypeptide obtained by        the method of embodiment 11 in an in vitro diagnostic test for        the detection of anti-Zika virus antibodies.    -   23. Use of a Zika virus polypeptide according to any of        embodiments 1 to 10 or of a Zika virus polypeptide obtained by        the method of embodiment 11 in an in vitro diagnostic test for        the detection of anti-Zika virus antibodies according to any of        the methods of embodiments 12 to 21.    -   24. A reagent kit for the detection of anti-Zika virus        antibodies, comprising a Zika virus polypeptide according to any        of embodiments 1 to 10 or of a Zika virus polypeptide obtained        by the method of embodiment 11.    -   25. A reagent kit according to embodiment 24 comprising in        separate containers or in separated compartments of a single        container unit at least microparticles coated with avidin or        streptavidin, and a polypeptide according to any of embodiments        1 to 10 or obtained by a method according to embodiment 11 that        is covalently coupled to biotin.    -   26. A reagent kit according to embodiment 24 comprising in        separate containers or in separated compartments of a single        container unit at least microparticles coated with one partner        of a bioaffine pair such as hapten/anti-hapten, and a        polypeptide according to any of embodiments 1 to 10 or obtained        by a method according to embodiment 11, wherein said bioaffine        pair is hapten/anti-hapten, in an embodiment        digoxin/anti-digoxin.    -   27. A reagent kit according to embodiment 24 additionally        comprising a second polypeptide according to any of embodiments        1 to 10 or obtained by a method according to embodiment 11 that        carries a detectable label.    -   28. A reagent kit according to embodiment 24, comprising in        separate containers or in separated compartments of a single        container unit at least microparticles coated with avidin or        streptavidin, and a μ-capture binding partner that is covalently        coupled to biotin.    -   29. A reagent kit according to embodiment 24, wherein said Zika        virus polypeptide carries a detectable label.    -   30. A method for detecting antibodies against a Zika virus in an        isolated biological sample presumed to contain antibodies        against at least one other non-Zika flavivirus, by using a Zika        virus NS1 polypeptide comprising a complete or partial sequence        of the ß-ladder domain as specific binding partner, wherein the        cross-reactivity against said non-Zika flavivirus is eliminated        by adding a polypeptide comprising the NS1 ß-ladder domain of        said Zika virus in an unlabeled form, in an embodiment adding        said polypeptide comprising the ß-ladder domain as a quencher.    -   31. A method according to embodiment 30, wherein said added        polypeptide comprising the Zika virus NS1 ß-ladder domain        consists essentially of SEQ ID NO:4, in an embodiment consists        of SEQ ID NO:4.    -   32. Use of Zika virus NS1 ß-ladder domain as a reagent for        reducing interference in an immunoassay for detecting anti-Zika        virus antibodies, in an embodiment said NS1 ß-ladder domain        consisting essentially of SEQ ID NO:4, in an embodiment        consisting of SEQ ID NO:4.

The invention is further illustrated by the Examples.

EXAMPLES Example 1: Immunological Reactivity (i.e., Antigenicity) ofDifferent Zika NS1 Antigen Variants in a Zika IgG Immunoassay

NS1 antigens and variants were essentially cloned, expressed, purifiedand labeled as described in WO2014054990A1 or by Scholz et al., J. Mol.Biol. (2005) 345, 1229-1241. The purified and solubilized gene productswere subsequently coupled to either biotin or to anelectrochemiluminescent ruthenium label.

The immunological reactivity (i.e., antigenicity) of the polypeptidefusion variants of Zika NS1 antigens was assessed in automated Elecsys®2010 and cobas e 411 analyzers (Roche Diagnostics GmbH). Elecsys® is aregistered trademark of the Roche group. Measurements were carried outin the double antigen sandwich format.

Signal detection in Elecsys® 2010 and cobas e 411 is based onelectrochemiluminescence. The biotin-conjugate (i.e. thecapture-antigen) is immobilized on the surface of a streptavidin coatedmagnetic bead whereas the detection-antigen bears a complexed Rutheniumcation (switching between the redox states 2+ and 3+) as the signalingmoiety. In the presence of a specific immunoglobulin analyte, thechromogenic ruthenium complex is bridged to the solid phase and emitslight at 620 nm after excitation at a platinum electrode. The signaloutput is in relative light units. Typically, the total duration of anassay is 18 minutes.

The recombinant Zika NS1 antigen fusion polypeptides were assessed in adouble antigen sandwich (DAGS) immunoassay format. To this end,recombinant Zika NS1 antigen was used as a biotin and a rutheniumconjugate pair, respectively, to detect anti-Zika NS1 antibodies inhuman sera.

NS1 is one of the immunodominant antigens of Zika Virus, and solublevariants of NS1 antigen—as disclosed in this patent application—areinvaluable tools for the detection of Zika virus infections. In allmeasurements, chemically polymerized and unlabeled SlyD-SlyD wasimplemented in large excess (˜8 μg/ml) in the reaction buffer asanti-interference substances to avoid immunological cross reactions viathe chaperone fusion and linker units.

In particular, two Zika NS1 variants were scrutinized in this study,namely the Zika NS1 “wing” domain (see SEQ ID NOs: 1 and 2) and the ZikaNS1 “ß-ladder” domain (see SEQ ID NO:4). In order to detect anti-ZikaNS1 IgG molecules, SlyD-SlyD-Zika NS1-biotin and SlyD-SlyD-ZikaNS1-ruthenium were used in R1 (reagent buffer 1) and R2 (reagent buffer2), respectively. The concentrations of the antigen conjugates in R1 andR2, respectively, were 500 ng/ml each.

Human serum samples negative for both Zika and Dengue IgG antibodies,human serum samples positive for Zika IgG antibodies and human serumsamples positive for Dengue IgG antibodies were tested with both of theZika NS1 recombinant antigens (“wing” and “ß-ladder”) in comparison witha commercially available state of the art immuno assay (indirect ELISAapplying a recombinant NS1 antigen coated to a microtiter plate,detection of the sample antibodies against Zika virus by addition of anenzyme-labeled anti-human IgG conjugate), as described by Huzly et al.,supra.

In this experiment, the human samples described above were assessed withthe aforementioned DAGS immunoassay setup.

The unavoidable system-inherent signal is around 500 counts. Lowbackground signals for human serum samples negative for Zika IgG andDengue IgG antibodies are indicative of high solubility and generallybenign physicochemical properties of the respective antigen rutheniumconjugates. Hydrophobic or, generally spoken, “sticky” antigen-rutheniumconjugates tend to interact with the bead surface and thereby increasethe background signal. From table 1 we can infer that thephysicochemical properties of Zika NS1 “wing” are excellent (column 1).This holds true for Zika NS1 “ß-ladder” as well (data column 2): WhenZika NS1 “wing” (SEQ ID NO:21) is used as an antigen pair inbiotinylated form and in ruthenylated form in the DAGS format, or ZikaNS1 “ß-ladder” (SEQ ID NO:22) is used as an antigen pair in biotinylatedand ruthenylated form in the DAGS format, both antigen pairs yield asignal background of ˜600-900 counts with negative human sera, whichclearly points to good solubility properties of the antigen conjugates.However, it becomes evident at first glance that the Zika NS1 “wing” andthe Zika NS1 “ß-ladder”, although being equivalent in their capabilityto detect anti-Zika antibodies, largely differ in their cross-reactivitywith anti-Dengue antibodies as shown in Table 1. Having a closer look atthe Zika IgG positive samples, we find that both Zika NS1 “wing” andZika NS1 “ß-ladder” detect all Zika IgG samples as positive (>2000counts). Importantly, Zika NS1 “wing” does not cross-react with DengueIgG positive samples, since all Dengue IgG samples are found negative(<<2000 counts) with the Zika NS1 wing antigen. In marked contrast, ZikaNS1 “ß-ladder” detects 9 out of 20 Dengue IgG samples as reactive (>2000counts), which points to a considerable degree of cross-reactivity. Avery similar reactivity pattern with the Zika IgG and the Dengue IgGpositive samples is observed with the commercially available Zika IgGassay described by Huzly et al. (Euro Surveill. 2016; 21(16), pii=30203,1-4), indicating that the commercially available Zika IgG assay suffersfrom considerable immunological cross-reactivity (i.e., the commerciallyavailable Zika IgG assay provokes quite a few false positive Zikaresults) with Dengue IgG positive samples. In conclusion, bothengineered variants of the Zika NS1 antigen (Zika NS1 “wing” and ZikaNS1 “ß-ladder”) possess outstanding physicochemical and antigenicproperties. Yet, the Zika NS1 “wing” antigen outperforms the ß-ladderdomain in that it displays superior specificity for Zika IgG antibodiesand significantly reduced immunological cross reactivity with Denguepositive sera.

The engineered recombinant Zika NS1 “wing” domain therefore constitutesa superior NS1 variant for specific determination of Zika antibodies ascompared to the state of the art commercially available Zika IgGimmunoassay (which presumably is based on the full-length NS1) asdescribed by Huzly et al., supra.

Table 1 shows the superior specificity (i.e., the strongly reducedcross-reactivity with anti-Dengue antibodies) of Zika NS1 “wing” ascompared to Zika NS1 “ß-ladder” and commercially available state of theart Zika IgG assay.

TABLE 1 Zika Zika NS1 “wing” Zika NS1 “ß-ladder” Commercial Zika NS1 NS1“ß- Commercial assay assay Zika IgG “wing” ladder” Zika IgG SignalSignal assay assay assay assay Sample ID Sample type counts dynamiccounts dynamics* s/co result result result BD03 Negative 1 562 0.33 9570.36 0.147 NR NR NR BD09 Negative 2 549 0.33 975 0.37 0.018 NR NR NRBD15 Negative 3 577 0.34 854 0.32 0.466 NR NR NR BD22 Negative 4 5590.33 874 0.33 0.056 NR NR NR BD23 Negative 5 557 0.33 879 0.33 0.034 NRNR NR BD24 Negative 6 567 0.34 869 0.33 0.050 NR NR NR BD29 Negative 7564 0.33 844 0.32 0.058 NR NR NR BD34 Negative 8 575 0.34 971 0.37 0.028NR NR NR BD36 Negative 9 546 0.32 799 0.30 0.010 NR NR NR BD37 Negative10 558 0.33 823 0.31 0.054 NR NR NR ARSZ16052 Zika IgG 28556 16.95 8012930.19 4.66 reactive reactive reactive ARSZ16244 Zika IgG 19906 11.8210274 3.87 3.65 reactive reactive reactive 16CDV61200 Zika IgG 9157 5.44113815 42.89 5.73 reactive reactive reactive ARSZ16245 Zika IgG 1799010.68 22353 8.42 4.89 reactive reactive reactive 16CDV61275 Zika IgG36305 21.56 241699 91.07 8.42 reactive reactive reactive ARSZ16271 ZikaIgG 27026 16.05 117869 44.41 7.46 reactive reactive reactive ARSZ16264Zika IgG 7260 4.31 45774 17.25 4.63 reactive reactive reactive ARSZ16178Zika IgG 27957 16.60 241364 90.95 6.12 reactive reactive reactiveARSZ16013 Zika IgG 9658 5.73 178153 67.13 6.49 reactive reactivereactive ARSZ16062 Zika IgG 21206 12.59 269333 101.49 7.66 reactivereactive reactive 8DEN0016 Dengue IgG 555 0.33 604 0.23 0.050 NR NR NR8DEN0008 Dengue IgG 558 0.33 616 0.23 0.213 NR NR NR 8DEN0040 Dengue IgG560 0.33 574 0.22 0.089 NR NR NR 8DEN0015 Dengue IgG 564 0.33 596 0.220.066 NR NR NR 8DEN0017 Dengue IgG 564 0.33 588 0.22 −0.019 NR NR NR8DEN0045 Dengue IgG 568 0.34 584 0.22 0.112 NR NR NR D117 Dengue IgG 5690.34 616 0.23 0.283 NR NR NR 8DEN0024 Dengue IgG 570 0.34 612 0.23 0.070NR NR NR 8DEN0023 Dengue IgG 572 0.34 675 0.25 0.054 NR NR NR 8DEN0041Dengue IgG 572 0.34 742 0.28 0.147 NR NR NR D092 Dengue IgG 566 0.344366 1.65 2.48 NR NR NR D096 Dengue IgG 567 0.34 4500 1.70 2.08 NRreactive reactive D009 Dengue IgG 574 0.34 19740 7.44 2.42 NR reactivereactive D094 Dengue IgG 577 0.34 2591 0.98 2.43 NR reactive reactiveD109 Dengue IgG 583 0.35 9702 3.66 1.71 NR reactive reactive D099 DengueIgG 585 0.35 3186 1.20 2.52 NR reactive reactive D102 Dengue IgG 5850.35 13330 5.02 1.30 NR reactive reactive 8DEN0029 Dengue IgG 587 0.3538740 14.60 3.62 NR reactive reactive 8DEN0013 Dengue IgG 595 0.35 3798014.31 3.46 NR reactive reactive 8DEN0007 Dengue IgG 601 0.36 11840 4.463.06 NR reactive reactive *Signal Dynamics = counts sample/((counts meannegative samples) × 3) NR = non-reactive

Example 2: Blocking Experiment to Corroborate the Superior Specificityof the Zika NS1 “Wing” Antigen as Compared to Zika NS1 “ß-Ladder”Antigen

The immunological reactivity (i.e., the antigenicity) of the polypeptidefusion variants of Zika NS1 antigens was assessed in automated Elecsys®2010 and cobas e 411 analyzers (Roche Diagnostics GmbH) as described inExample 1.

Three human serum samples positive for Zika IgG antibodies were testedwith both of the engineered Zika NS1 recombinant antigens (“wing” and“ß-ladder”). In parallel, these human serum samples positive for ZikaIgG antibodies were individually spiked with full-length flavivirus NS1antigen preparations of either TBEV (tick-borne encephalitis virus,FSME, SEQ ID NO:6), or one antigen of DENV1-4 (Dengue virus 1-4, SEQ IDNOs:8, 10, 12, 14), or WNV (West-Nile virus, SEQ ID NO:16), or YFV(yellow fever virus, SEQ ID NO:18), or JEV (Japanese encephalitis virus,SEQ ID NO:20) or ZIKV (Zika virus, SEQ ID NO:3) (tables 2a and 2b).

In this experiment, the human samples (spiked and unspiked) as describedabove were assessed with the aforementioned DAGS immunoassay setup.

Samples were pre-diluted to a reactivity level (titer) needed for theblocking experiment. This step was needed as the concentration of theflavivirus NS-1 preparations prepared for the blocking experiment andthe amount of antibodies in the sample need to be within a reasonableconcentration ratio in order to yield a clear blocking result. Then thereactivity of the pre-diluted human anti-Zika IgG positive sample wascompared to the signals achieved with the same sample when spiked withfull-length flavivirus NS-1 of either TBEV, or DENV1-4, or WNV, or YFV,or JEV or ZIKV. The extent of signal reduction due to competition by thedifferent full-length flavivirus NS-1 preparations of TBEV, DENV1-4,WNV, YFV, JEV and ZIKV was calculated. The signal reduction/extent ofblocking was normalized to the maximal blocking achieved with fulllength Zika NS1, which, as expected, shows the strongest quenching ofthe signal with both assays (irrespective of the use of either Zika NS1“wing” or Zika NS1 “ß-ladder”). The degree of the capacity to competewas calculated for all other full-length flavivirus NS1 preparations(TBEV, DENV1-4, WNV, YFV, JEV). It is evident at first glance that thesignal of the assay based on the Zika NS1 “wing” antigen is only weaklyquenched by the non-Zika NS1 antigens, whereas the signal of the assaybased on the Zika NS1 “ß-ladder” antigen is markedly reduced by TBEV,DENV1-4 and JEV. This finding compellingly indicates that the NS1 wingdomain and the NS1 ß-ladder domain strongly differ in their structuraluniqueness among the arboviral NS1 homologues. Obviously, the wing partof the non-Zika NS1 antigens is not able to efficiently compete with theengineered Zika wing antigen for binding to the anti-Zika analyteantibodies. Conversely, the ß-ladder part of the non-Zika NS1 antigensis perfectly able to efficiently compete with the engineered Zikaß-ladder antigen for binding to the anti-Zika analyte antibodies.

In conclusion, this experiment is perfectly in line with Example 1 as italso demonstrates that the assay based on Zika NS1 “wing” antigenlargely detects those anti-Zika IgG antibodies, which are notcross-reactive (or prone to cross-reactivity) with flavivirus NS-1preparations of other arboviruses (TBEV, DENV1-4, WNV, YFV, JEV). Incontrast, the assay based on Zika NS1 “ß-ladder” antigen binds anti-Zikaantibodies, which are, in part, cross-reactive (or prone tocross-reactivity) with TBEV, DENV1-4 and JEV (i.e., they can be blockedby TBEV, DENV1-4 and JEV NS-1 preparations). In other words, theß-ladder domain of the Zika NS1 antigen seems to share significantstructural and sequence homology with the ß-ladder domains of relatedarboviruses, thereby evoking false positive results in immunoassays whenused as an antigen. In contrast, the wing domain of the Zika NS1 antigenseems to be rather unique and seems to share little structural homologywith its NS counterparts from other arboviruses.

Our data indicate that this principle holds true not only in thediscrimination of Zika virus infections from other flaviviralinfections, but also in the discrimination of other flaviviralinfections from each other. We conclude that the specific detection ofantibodies against the wing domain of the NS1 antigen may be anexcellent means to clearly distinguish flaviviral infections even on amultiple flaviviral infection background. We reason that our findingsbear promise to markedly improve the current serology and to obtain amore appropriate view on prevalence and incidence data in the flavivirusfield.

In summary, the Zika NS1 “wing” antigen displays outstanding andsuperior specificity for Zika IgG, is less susceptible to immunologicalcross-reactivity with antibodies raised against NS1 homologues fromother arboviruses (here: family of flaviviruses) and is therefore muchmore suitable for specific testing for anti-Zika IgG antibodies.

TABLE 2a SS-ZIKA-NS1- S-ZIKA-NS1- SS-ZIKA-NS1- S-ZIKA-NS1- SS-ZIKA-NS1-S-ZIKA-NS1- wing ß-ladder wing ß-ladder wing ß-ladder NormalizedNormalized to Sample 16CDV61275 counts counts Blocking [%] Blocking [%]to Zika Zika 1:10 pre-diluted 3108 3432 Blocking [%] Blocking [%] Spikedwith ZIKV NS1 1225 947 76.3 92.0 100.0 100.0 Spiked with TBEV NS1 30643387 1.73 1.62 2.3 1.8 Spiked with DenV1 NS1 2946 1511 6.38 69.1 8.475.2 Spiked with DenV2 NS1 3042 3349 2.60 2.99 3.4 3.2 Spiked with DenV3NS1 3014 1587 3.70 66.4 4.9 72.2 Spiked with DenV4 NS1 3045 2734 2.4825.1 3.3 27.3 Normalized Normalized to Sample 16CDV61278 counts countsBlocking [%] Blocking [%] to Zika Zika 1:20 pre-diluted 2989 2887Blocking [%] Blocking [%] Spiked with ZIKV NS1 759 1457 91.7 69.4 100.0100.0 Spiked with TBEV NS1 2914 2911 3.10 −1.07 3.4 −1.5 Spiked withDenV1 NS1 2858 1115 5.41 79.3 5.9 114.3 Spiked with DenV2 NS1 2749 14239.91 65.5 10.8 94.4 Spiked with DenV3 NS1 2783 1695 8.51 53.4 9.3 76.9Spiked with DenV4 NS1 2880 2829 4.50 2.60 4.9 3.7 Normalized Normalizedto Sample 16CDV61197 counts counts Blocking [%] Blocking [%] to ZikaZika 1:10 pre-diluted 2925 3089 Blocking [%] Blocking [%] Spiked withZIKV NS1 1127 1720 76.1 62.8 100.0 100.0 Spiked with TBEV NS1 2897 30451.19 1.81 1.6 2.9 Spiked with DenV1 NS1 2648 2073 11.7 41.7 15.4 66.4Spiked with DenV2 NS1 2682 2013 10.3 44.2 13.5 70.4 Spiked with DenV3NS1 2666 1911 11.0 48.4 14.4 77.0 Spiked with DenV4 NS1 2904 2320 0.89131.6 1.2 50.3

TABLE 2b SS-ZIKA-NS1- S-ZIKA-NS1- SS-ZIKA-NS1- S-ZIKA-NS1- SS-ZIKA-NS1-S-ZIKA-NS1- wing ß-ladder wing ß-ladder wing ß-ladder NormalizedNormalized Sample 16CDV61275 counts counts Blocking [%] Blocking [%] toZika to Zika 1:10 pre-diluted 3280 3486 Blocking [%] Blocking [%] Spikedwith ZIKV NS1 920 825 86.7 94.2 100.0 100.0 Spiked with WNV NS1 25962154 25.1 47.1 29.0 50.1 Spiked with YFV NS1 3154 3257 4.63 8.10 5.3 8.6Spiked with JEV NS1 3095 3198 6.80 10.2 7.8 10.8 Normalized NormalizedSample 16CDV61278 counts counts Blocking [%] Blocking [%] to Zika toZika 1:20 pre-diluted 2920 3314 Blocking [%] Blocking [%] Spiked withZIKV NS1 1098 1601 77.1 64.5 100.0 100.0 Spiked with WNV NS1 2711 24778.85 31.5 11.5 48.9 Spiked with YFV NS1 2651 3093 11.4 8.33 14.8 12.9Spiked with JEV NS1 2885 2835 1.48 18.0 1.9 28.0 Normalized NormalizedSample 16CDV61197 counts counts Blocking [%] Blocking [%] to Zika toZika 1:10 pre-diluted 2745 2989 Blocking [%] Blocking [%] Spiked withZIKV NS1 1403 1528 61.3 62.7 100.0 100.0 Spiked with WNV NS1 2626 25515.44 18.8 8.9 30.0 Spiked with YFV NS1 2664 2844 3.70 6.23 6.0 9.9Spiked with JEV NS1 2549 2704 8.96 12.2 14.6 19.5

Explanation of Acronyms

ZIKV=Zika virus

DENV1=Dengue Virus Type 1 DENV2=Dengue Virus Type 2 DENV3=Dengue VirusType 3 DENV4=Dengue Virus Type 4 WNV=West Nile Virus JEV=JapaneseEncephalitis Virus YFV=Yellow Fever Virus TBEV=Tick Borne EncephalitisVirus (=FSME Virus)

1. A polypeptide suitable for detecting antibodies against Zika virus inan isolated biological sample comprising a Zika virus NS1 wing domainspecific amino acid sequence, wherein no amino acid sequences from theZika virus NS1 ß-ladder domain are present in said polypeptide.
 2. Apolypeptide according to claim 1, wherein said Zika virus NS1 wingdomain specific amino acid sequence comprises SEQ ID NO. 1 or 2 andwherein no amino acid sequences from the Zika virus NS1 ß-ladder domainare present in said polypeptide.
 3. A polypeptide according to claim 1,wherein no further Zika virus specific amino acid sequences are presentin said polypeptide.
 4. A polypeptide according to claim 1 wherein saidZika polypeptide is fused to a chaperone.
 5. A polypeptide according toclaim 1 wherein said chaperone is selected from the group consisting ofSlyD, SlpA, FkpA and Skp.
 6. A polypeptide according to claim 5consisting of SEQ ID NO:21.
 7. A polypeptide according to claim 1,wherein in an immunoassay for detecting antibodies against Zika virus,said Zika NS1 polypeptide does not immunologically cross-react withantibodies raised against structurally related antigens from tick-borneencephalitis virus comprising any of SEQ ID NOs:5 or 6, and/or fromDengue virus 1-4 comprising any of SEQ ID NOs:7 to 14, and/or from WestNile virus comprising any of SEQ ID NOs:15 or 16, and/or from yellowfever virus comprising any of SEQ ID NOs:17 or 18, and/or from Japaneseencephalitis virus comprising any of SEQ ID NOs:19 to 20, butimmunologically reacts with antibodies raised against full length Zikavirus NS1 polypeptide according to SEQ ID NO:3.
 8. A method of producinga soluble and immunoreactive Zika virus NS1 wing domain polypeptide,said method comprising the steps of a) culturing host cells transformedwith an expression vector comprising operably linked a recombinant DNAmolecule encoding a Zika virus NS1 wing domain polypeptide according toclaim 1, b) expression of said Zika virus NS1 polypeptide and c)purification of said Zika virus NS1 polypeptide.
 9. A method fordetecting antibodies specific for Zika virus in an isolated sample saidmethod comprising a) forming an immunoreaction admixture by admixing abody fluid sample with a Zika virus polypeptide according to claim 1; b)maintaining said immunoreaction admixture for a time period sufficientfor allowing antibodies present in the body fluid sample against saidZika virus polypeptide to immunoreact with said Zika virus polypeptideto form an immunoreaction product; and c) detecting the presence and/orthe concentration of any of said immunoreaction product.
 10. The methodfor detecting antibodies specific for Zika virus in an isolated sampleaccording to claim 9, wherein the detected antibody is an IgG or IgMantibody.